RESUMEN
We have reported recently that inactivation of the essential autophagy-related gene 7 (Atg7) in keratinocytes has little or no impact on morphology and function of the epidermal barrier in experimental animals. When these mice aged, mutant males, (Atg7 ΔKC), developed an oily coat. As the keratin 14 promoter driven cre/LoxP system inactivates floxed Atg7 in all keratin 14 (K14) expressing cells, including sebocytes, we investigated whether the oily hair phenotype was the consequence of changes in function of the skin sebaceous glands. Using an antibody to the GFP-LC3 fusion protein, autophagosomes were detected at the border of sebocyte disintegration in control but not in mutant animals, suggesting that autophagy was (a) active in normal sebaceous glands and (b) was inactivated in the mutant mice. Detailed analysis established that dorsal sebaceous glands were about twice as large in all Atg7 ΔKC mice compared to those of controls (Atg7 F/F), and their rate of sebocyte proliferation was increased. In addition, male mutant mice yielded twice as much lipid per unit hair as age-matched controls. Analysis of sebum lipids by thin layer chromatography revealed a 40% reduction in the proportion of free fatty acids (FFA) and cholesterol, and a 5-fold increase in the proportion of fatty acid methyl esters (FAME). In addition, the most common diester wax species (58-60 carbon atoms) were increased, while shorter species (54-55 carbon atoms) were under-represented in mutant sebum. Our data show that autophagy contributes to sebaceous gland function and to the control of sebum composition.
Asunto(s)
Proteína 7 Relacionada con la Autofagia/genética , Autofagia/genética , Glándulas Sebáceas/patología , Glándulas Sebáceas/fisiopatología , Sebo/química , Animales , Autofagosomas , Proliferación Celular/genética , Colesterol/análisis , Ácidos Grasos no Esterificados/análisis , Cabello , Masculino , Ratones , Fenotipo , Ceras/análisisRESUMEN
OBJECTIVE: Cingulin is a cytoplasmic component of tight junctions. Although modulation of cingulin levels in cultured epithelial model systems has no significant effect on barrier function, evidence from cingulin knockout mice suggests that cingulin may be involved in the regulation of the behavior of epithelial or endothelial cells. Here, we investigate the role of cingulin in the barrier function of endothelial cells. APPROACH AND RESULTS: We show that cingulin is expressed in human endothelial cells of the skin, brain, and lung in vivo and in vitro. Endothelial cingulin colocalizes and coimmunoprecipitates with the tight junction proteins zonula occludens-1 and guanine nucleotide exchange factor-H1. Cingulin overexpression in human umbilical vein endothelial cell induces tight junction formation, increases transendothelial electric resistance, and strengthens barrier function for low and high molecular weight tracers. In contrast, cultured endothelial cells lacking cingulin are more permeable for low molecular weight tracers. In cingulin knockout mice, neurons of the area postrema and Purkinje cells show an increased uptake of small molecular weight tracers indicating decreased barrier function at these sites. CONCLUSIONS: We demonstrate that cingulin participates in the modulation of endothelial barrier function both in human cultured cells in vitro and in mouse brains in vivo. Understanding the role of cingulin in maintaining tight barriers in endothelia may allow developing new strategies for the treatment of vascular leak syndromes.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Permeabilidad Capilar , Células Endoteliales/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Animales , Área Postrema/metabolismo , Proliferación Celular , Células Cultivadas , Claudina-5/metabolismo , Impedancia Eléctrica , Genotipo , Humanos , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Fenotipo , Células de Purkinje/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Uniones Estrechas/metabolismo , Factores de Tiempo , Transfección , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
BACKGROUND: Prosthetic implant infections caused by Staphylococcus aureus and epidermidis are major challenges for early diagnosis and treatment owing to biofilm formation on the implant surface. Extracellular DNA (eDNA) is actively excreted from bacterial cells in biofilms, contributing to biofilm stability, and may offer promise in the detection or treatment of such infections. QUESTIONS/PURPOSES: (1) Does DNA structure change during biofilm formation? (2) Are there time-dependent differences in eDNA production during biofilm formation? (3) Is there differential eDNA production between clinical and control Staphylococcal isolates? (4) Is eDNA production correlated to biofilm thickness? METHODS: We investigated eDNA presence during biofilm formation in 60 clinical and 30 control isolates of S aureus and S epidermidis. The clinical isolates were isolated from patients with infections of orthopaedic prostheses and implants: 30 from infected hip prostheses and 30 from infected knee prostheses. The control isolates were taken from healthy volunteers who had not been exposed to antibiotics and a hospital environment during the previous 3 and 12 months, respectively. Control S epidermidis was isolated from the skin of the antecubital fossa, and control S aureus was isolated from the nares. For the biofilm experiments the following methods were used to detect eDNA: (1) fluorescent staining with 4',6-diamidino-2-phenylindole (DAPI), (2) eDNA extraction using a commercial kit, and (3) confocal laser scanning microscopy for 24-hour biofilm observation using propidium iodide and concanavalin-A staining; TOTO®-1 and SYTO® 60 staining were used for observation and quantification of eDNA after 6 and 24 hours of biofilm formation. Additionally antibiotic resistance was described. RESULTS: eDNA production as observed by confocal laser scanning microscopy was greater in clinical isolates than controls (clinical isolates mean ± SD: 1.84% ± 1.31%; control mean ± SD: 1.17% ± 1.37%; p < 0.005) after 6 hours of biofilm formation. After 24 hours, the amount of eDNA was greater in biofilms of S epidermidis than in biofilms of S aureus (S aureus mean ± SD: 1.35% ± 2.0%; S epidermidis mean ± SD: 6.42% ± 10.6%; p < 0.05). Clinical isolates of S aureus and S epidermidis produced more eDNA than control isolates at 6 hours of biofilm formation. The extraction method also showed that clinical isolates produced substantially greater amounts of eDNA than controls. CONCLUSIONS: S aureus and S epidermidis exhibit a differential production of DNA with time. Clinical isolates associated with implant infections produce greater amounts of eDNA than controls. Future research might focus on the diagnostic value of eDNA as a surrogate laboratory marker for biofilm formation in implant infections. CLINICAL RELEVANCE: eDNA should be considered as a potential future diagnostic tool or even a possible target to modify biofilms for successful treatment of biofilm-associated infections.
Asunto(s)
Biopelículas , ADN Bacteriano/análisis , Espacio Extracelular/genética , Infecciones Relacionadas con Prótesis/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus/aislamiento & purificación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Prótesis de Cadera/microbiología , Humanos , Prótesis de la Rodilla/microbiología , Masculino , Persona de Mediana Edad , Staphylococcus/genética , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/aislamiento & purificación , Adulto JovenRESUMEN
INTRODUCTION: Skeletal muscle consists of different fiber types which adapt to exercise, aging, disease, or trauma. Here we present a protocol for fast staining, automatic acquisition, and quantification of fiber populations with ImageJ. METHODS: Biceps and lumbrical muscles were harvested from Sprague-Dawley rats. Quadruple immunohistochemical staining was performed on single sections using antibodies against myosin heavy chains and secondary fluorescent antibodies. Slides were scanned automatically with a slide scanner. Manual and automatic analyses were performed and compared statistically. RESULTS: The protocol provided rapid and reliable staining for automated image acquisition. Analyses between manual and automatic data indicated Pearson correlation coefficients for biceps of 0.645-0.841 and 0.564-0.673 for lumbrical muscles. Relative fiber populations were accurate to a degree of ± 4%. CONCLUSIONS: This protocol provides a reliable tool for quantification of muscle fiber populations. Using freely available software, it decreases the required time to analyze whole muscle sections. Muscle Nerve 54: 292-299, 2016.
Asunto(s)
Fibras Musculares Esqueléticas/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Animales , Diagnóstico por Computador , Diagnóstico por Imagen , Inmunohistoquímica , Masculino , Cadenas Pesadas de Miosina/clasificación , Ratas , Ratas Sprague-Dawley , Análisis de RegresiónRESUMEN
Drebrin an actin-bundling key regulator of dendritic spine genesis and morphology, has been recently proposed as a regulator of hippocampal glutamatergic activity which is critical for memory formation and maintenance. Here, we examined the effects of genetic deletion of drebrin on dendritic spine and on the level of complexes containing major brain receptors. To this end, homozygous and heterozygous drebrin knockout mice generated in our laboratory and related wild-type control animals were studied. Level of protein complexes containing dopamine receptor D1/dopamine receptor D2, 5-hydroxytryptamine receptor 1A (5-HT1(A)R), and 5-hydroxytryptamine receptor 7 (5-HT7R) were significantly reduced in hippocampus of drebrin knockout mice whereas no significant changes were detected for GluR1, 2, and 3 and NR1 as examined by native gel-based immunoblotting. Drebrin depletion also altered dendritic spine formation, morphology, and reduced levels of dopamine receptor D1 in dendritic spines as evaluated using immunohistochemistry/confocal microscopy. Electrophysiological studies further showed significant reduction in memory-related hippocampal synaptic plasticity upon drebrin depletion. These findings provide unprecedented experimental support for a role of drebrin in the regulation of memory-related synaptic plasticity and neurotransmitter receptor signaling, offer relevant information regarding the interpretation of previous studies and help in the design of future studies on dendritic spines.
Asunto(s)
Espinas Dendríticas/fisiología , Hipocampo/fisiología , Memoria/fisiología , Plasticidad Neuronal/fisiología , Neuropéptidos/metabolismo , Receptores de Neurotransmisores/metabolismo , Animales , Western Blotting , Potenciales Postsinápticos Excitadores/fisiología , Inmunohistoquímica , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Técnicas de Placa-ClampRESUMEN
In contextual fear conditioning (CFC), the use of pharmacological and lesion approaches has helped to understand that there are differential roles for the dorsal hippocampus (DH) and the ventral hippocampus (VH) in the acquisition, consolidation and retrieval phases. Concomitant analysis of the DH and the VH in individual phases with respect to α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors and N-methyl-D-aspartate receptor subtype N1 (GluN1)-containing complexes (RCC) and subunits has not been reported so far. Herein, CFC was performed in mice that were euthanized at different time points. DH and VH samples were taken for the determination of RCC and subunit levels using BN- and SDS-PAGE, respectively, with subsequent Western blotting. Evaluation of spine densities, morphology, and immunohistochemistry of GluA1 and GluA2 was performed. In the acquisition phase levels of GluA1-RCC and subunits in VH were increased. In the consolidation phase GluA1- and GluA2-RCC levels were increased in DH and VH, while both receptor subunit levels were increased in the VH only. In the retrieval phase GluA1-RCC, subunits thereof and GluA2-RCC were increased in DH and VH, whereas GluA2 subunits were increased in the VH only. GluN1-RCC levels were increased in acquisition and consolidation phase, while subunit levels in the acquisition phase were increased only in the DH. The immunohistochemical studies in the individual phases in subareas of hippocampus supported immunochemical changes of GluA1 and GluA2 RCC's. Dendritic spine densities and the prevalence of thin spines in the acquisition phase of VH and mushroom spines in the retrieval phase of the VH and DH were increased. The findings from the current study suggest different receptor and receptor complex patterns in the individual phases in CFC and in DH and VH. The results propose that different RCCs are formed in the individual phases and that VH and DH may be involved in CFC.
Asunto(s)
Condicionamiento Psicológico/fisiología , Miedo/fisiología , Hipocampo/metabolismo , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Conducta Espacial/fisiología , Animales , Far-Western Blotting , Cromatografía Liquida , Espinas Dendríticas/metabolismo , Electroforesis en Gel de Poliacrilamida , Electrochoque , Hipocampo/citología , Inmunohistoquímica , Inmunoprecipitación , Masculino , Espectrometría de Masas , Memoria/fisiología , Ratones Endogámicos C57BL , Pruebas NeuropsicológicasRESUMEN
Interleukin-33 (IL-33) is a recently described member of the IL-1 family of cytokines, which was identified as a ligand for the ST2 receptor. Components of the IL-33/ST2 system were shown to be expressed in normal and pressure overloaded human myocardium, and soluble ST2 (sST2) has emerged as a prognostic biomarker in myocardial infarction and heart failure. However, expression and regulation of IL-33 in human adult cardiac myocytes and fibroblasts was not tested before. In this study we found that primary human adult cardiac fibroblasts (HACF) and human adult cardiac myocytes (HACM) constitutively express nuclear IL-33 that is released during cell necrosis. Tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-1ß significantly increased both IL-33 protein and IL-33 mRNA expression in HACF and HACM as well as in human coronary artery smooth muscle cells (HCASMC). The nuclear factor-κB (NF-κB) inhibitor dimethylfumarate inhibited TNF-α- and IL-1ß-induced IL-33 production as well as nuclear translocation of p50 and p65 NF-κB subunits in these cells. Mitogen-activated protein/extracellular signal-regulated kinase inhibitor U0126 abrogated TNF-α-, IFN-γ-, and IL-1ß-induced and Janus-activated kinase inhibitor I reduced IFN-γ-induced IL-33 production. We detected IL-33 mRNA in human myocardial tissue from patients undergoing heart transplantation (n=27) where IL-33 mRNA levels statistically significant correlated with IFN-γ (r=0.591, p=0.001) and TNF-α (r=0.408, p=0.035) mRNA expression. Endothelial cells in human heart expressed IL-33 as well as ST2 protein. We also reveal that human cardiac and vascular cells have different distribution patterns of ST2 isoforms (sST2 and transmembrane ST2L) mRNA expression and produce different amounts of sST2 protein. Both human macrovascular (aortic and coronary artery) and heart microvascular endothelial cells express specific mRNA for both ST2 isoforms (ST2L and sST2) and are a source for sST2 protein, whereas cardiac myocytes, cardiac fibroblasts and vascular SMC express only minor amounts of ST2 mRNA and do not secrete detectable amounts of sST2 antigen. In accordance with the cellular distribution of ST2 receptor, human cardiac fibroblasts and myocytes as well as HCASMC did not respond to treatment with IL-33, as recombinant human IL-33 did not induce NF-κB p50 and p65 subunits nuclear translocation or increase IL-6, IL-8, and monocyte chemoattractant protein (MCP-1) level in HACF, HACM and HCASMC. In summary, we found that endothelial cells seem to be the source of sST2 and the target for IL-33 in the cardiovascular system. IL-33 is expressed in the nucleus of human adult cardiac fibroblasts and myocytes and released during necrosis. Proinflammatory cytokines TNF-α, IFN-γ and IL-1ß increase IL-33 in these cells in vitro, and IL-33 mRNA levels correlated with TNF-α and IFN-γ mRNA expression in human myocardial tissue.
Asunto(s)
Vasos Coronarios/metabolismo , Fibroblastos/metabolismo , Regulación de la Expresión Génica/fisiología , Interleucinas/biosíntesis , Músculo Liso Vascular/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptores de Superficie Celular/biosíntesis , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Adulto , Butadienos/farmacología , Núcleo Celular/metabolismo , Células Cultivadas , Vasos Coronarios/citología , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/citología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mediadores de Inflamación/metabolismo , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Musculares/metabolismo , Músculo Liso Vascular/citología , Miocardio/citología , Miocitos Cardíacos/citología , Miocitos del Músculo Liso/citología , Subunidad p50 de NF-kappa B/metabolismo , Nitrilos/farmacología , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/fisiología , ARN Mensajero/biosíntesis , Factor de Transcripción ReIA/metabolismoRESUMEN
Systemic mastocytosis is a neoplastic disease of mast cells harboring the activating KIT mutation D816V. In most patients, mast cell infiltration in the bone marrow is accompanied by marked microenvironment alterations, including increased angiogenesis, osteosclerosis, and sometimes fibrosis. Little is known about the mast cell-derived molecules contributing to these bone marrow alterations. We show here that neoplastic mast cells in patients with systemic mastocytosis express oncostatin M (OSM), a profibrogenic and angiogenic modulator. To study the regulation of OSM expression, KIT D816V was inducibly expressed in Ba/F3 cells and was found to up-regulate OSM mRNA and protein levels, suggesting that OSM is a KIT D816V-dependent mediator. Correspondingly, KIT D816V(+) HMC-1.2 cells expressed significantly higher amounts of OSM than the KIT D816V(-) HMC-1.1 subclone. RNA interference-induced knockdown of STAT5, a key transcription factor in KIT D816V(+) mast cells, inhibited OSM expression in HMC-1 cells, whereas a constitutively activated STAT5 mutant induced OSM expression. Finally, OSM secreted from KIT D816V(+) mast cells stimulated growth of endothelial cells, fibroblasts, and osteoblasts, suggesting that mast cell-derived OSM may serve as a key modulator of the marrow microenvironment and thus contribute to the pathology of systemic mastocytosis.
Asunto(s)
Médula Ósea/patología , Mastocitosis Sistémica/metabolismo , Mastocitosis Sistémica/patología , Oncostatina M/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Western Blotting , Médula Ósea/metabolismo , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Expresión Génica , Regulación de la Expresión Génica/fisiología , Humanos , Immunoblotting , Inmunohistoquímica , Mastocitos/metabolismo , Mastocitos/patología , Mastocitosis Sistémica/genética , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT5/metabolismoRESUMEN
OBJECTIVE: Interleukin (IL)-33 is the most recently described member of the IL-1 family of cytokines and it is a ligand of the ST2 receptor. While the effects of IL-33 on the immune system have been extensively studied, the properties of this cytokine in the cardiovascular system are much less investigated. Methods/Results- We show here that IL-33 promoted the adhesion of human leukocytes to monolayers of human endothelial cells and robustly increased vascular cell adhesion molecule-1, intercellular adhesion molecule-1, endothelial selectin, and monocyte chemoattractant protein-1 protein production and mRNA expression in human coronary artery and human umbilical vein endothelial cells in vitro as well as in human explanted atherosclerotic plaques ex vivo. ST2-fusion protein, but not IL-1 receptor antagonist, abolished these effects. IL-33 induced translocation of nuclear factor-κB p50 and p65 subunits to the nucleus in human coronary artery endothelial cells and human umbilical vein endothelial cells and overexpression of dominant negative form of IκB kinase 2 or IκBα in human umbilical vein endothelial cells abolished IL-33-induced adhesion molecules and monocyte chemoattractant protein-1 mRNA expression. We detected IL-33 and ST2 on both protein and mRNA level in human carotid atherosclerotic plaques. CONCLUSIONS: We hypothesize that IL-33 may contribute to early events in endothelial activation characteristic for the development of atherosclerotic lesions in the vessel wall, by promoting adhesion molecules and proinflammatory cytokine expression in the endothelium.
Asunto(s)
Moléculas de Adhesión Celular/biosíntesis , Células Endoteliales/fisiología , Inflamación/etiología , Interleucinas/fisiología , Placa Aterosclerótica/etiología , Adhesión Celular , Células Cultivadas , Humanos , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Leucocitos/fisiología , FN-kappa B/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Receptores de Superficie Celular/fisiologíaRESUMEN
In the event of a myocardial infarction, current interventions aim to reopen the occluded vessel to reduce myocardial damage and injury. Although reperfusion is essential for tissue salvage, it can cause further damage and the onset of inflammation. We show a novel anti-inflammatory effect of a fibrin-derived peptide, Bbeta15-42. This peptide competes with the fibrin fragment N-terminal disulfide knot-II (an analog of the fibrin E1 fragment) for binding to vascular endothelial (VE)-cadherin, thereby preventing transmigration of leukocytes across endothelial cell monolayers. In acute or chronic rat models of myocardial ischemia-reperfusion injury, Bbeta15-42 substantially reduces leukocyte infiltration, infarct size and subsequent scar formation. The pathogenic role of fibrinogen products is further confirmed in fibrinogen knockout mice, in which infarct size was substantially smaller than in wild-type animals. Our findings conclude that the interplay of fibrin fragments, leukocytes and VE-cadherin contribute to the pathogenesis of myocardial damage and reperfusion injury. The naturally occurring peptide Bbeta15-42 represents a potential candidate for reperfusion therapy in humans.
Asunto(s)
Cadherinas/metabolismo , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/prevención & control , Fragmentos de Péptidos/metabolismo , Animales , Antígenos CD , Endotelio Vascular/citología , Productos de Degradación de Fibrina-Fibrinógeno/farmacología , Productos de Degradación de Fibrina-Fibrinógeno/uso terapéutico , Humanos , Masculino , Ratones , Infarto del Miocardio/tratamiento farmacológico , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/uso terapéutico , RatasRESUMEN
During tissue regeneration, mesenchymal stem cells can support endothelial cells in the process of new vessel formation. For a functional interaction of endothelial cells with mesenchymal stem cells a vascular inductive microenvironment is required. Using a cellular model for neo-vessel formation, we could show that newly formed vascular structures emanated from the embedded aggregates, consisting of mesenchymal stem cells co-cultured with autologous human umbilical vein endothelial cells, into avascular human platelet lysate-based matrices, bridging distances up to 5 mm to join with adjacent aggregates with the same morphology forming an interconnected network. These newly formed vascular sprouts showed branch points and generated a lumen, as sign of mature vascular development. In two-dimensional culture, we detected binding of mesenchymal stem cells to laser-damaged endothelial cells under flow conditions, mimicking the dynamics in blood vessels. In conclusion, we observed that mesenchymal stem cells can support human umbilical vein endothelial cells in their vitality and functionality. In xeno-free human platelet lysate-based matrices, endothelial cells form complex vascular networks in a primarily avascular scaffold with the aid of mesenchymal stem cells, when co-cultured in three-dimensional spherical aggregates. Under dynamic conditions, representing the flow rate of venous vessel, mesenchymal stem cells preferably bind to damaged endothelial cells presumably assisting in the healing process.
Asunto(s)
Células Madre Mesenquimatosas , Neovascularización Fisiológica , Humanos , Técnicas de Cocultivo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células CultivadasRESUMEN
A high throughput method based on flow injection analysis was developed and validated for the quantification of the peptide Bß(15-42) in cellular samples comparing different labeling strategies and detection methods. The used labels were 1,4,7,10-tetraazacyclododecane-N, N', N'', N'''-tetraaceticacid (In-DOTA) and 2-(4-isothiocyanatobenzyl) - 1,4,7,10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid (In-DOTA-Bn) for elemental labeling. 6-Hydroxy-9-(2-carboxyphenyl)- (3H)-xanthen-3-on (fluorescein) was employed as fluorescence label. The explored peptide (mass = 3 kD) is a novel candidate drug, which shows an anti-inflammatory effect after an event of myocardial infarction. The analysed samples were fractioned cell compartments of human umbilical cord vein endothelial cells (HUVEC) maintained via lysis with Triton X buffer. In order to enhance sensitivity and selectivity of peptide quantification via flow injection the peptide was labeled prior to incubation using elemental and fluorescence labels. Quantification of the elemental and fluorescence labeled peptide was performed via flow injection analysis combined with inductive coupled plasma sector field mass spectrometry (FIA-ICP-SFMS) or fluorescence detection (FIA-FLD), respectively. The employed quantification strategies were external calibration in the case of fluorescence detection and external calibration with and without internal standardization and on-line IDMS in the case of ICP-MS detectionThe limit of detection (LOD) for FIA-ICP-MS was 9 pM In-DOTA-Bß(15-42) (0.05 fmol absolute) whereas FIA-FLD showed a LOD of 100 pM (3 fmol absolute) for the fluorescein labeled peptide. Short term precision of FIA-ICP-MS was superior for all ICP-MS based quantification strategies compared to FIA-FLD (FIA-ICP-SFMS: 0.3-3.3%; FIA-FLD: 6.5%). Concerning long term precision FIA-ICP-SFMS with on-line IDMS and internal standardization showed the best results (3.1 and 4.6%, respectively) whereas the external calibration of both applied methodological approaches was only in the range of 10 %.The concentrations in the Triton X soluble fraction relative to the applied amount of Indium in the cell culture were in the range of 0.75-1.8% for In-DOTA or 0.30-0.79% for the 2-(4-isothiocyanatobenzyl) - 1,4,7,10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid (In-DOTA-Bn) labeled peptide Bß(15-42). In the Triton X insoluble fraction the relative concentrations of Indium were 0.03-0.18% for the In-DOTA labeled peptide and 0.03-0.13% for Bß(15-42)-In-DOTA-Bn.
RESUMEN
Obesity and body fat distribution are important risk factors for the development of type 2 diabetes and metabolic syndrome. Evidence has accumulated that this risk is related to intrinsic differences in behavior of adipocytes in different fat depots. We recently identified LIM domain only 3 (LMO3) in human mature visceral adipocytes; however, its function in these cells is currently unknown. The aim of this study was to determine the potential involvement of LMO3-dependent pathways in the modulation of key functions of mature adipocytes during obesity. Based on a recently engineered hybrid rAAV serotype Rec2 shown to efficiently transduce both brown adipose tissue (BAT) and white adipose tissue (WAT), we delivered YFP or Lmo3 to epididymal WAT (eWAT) of C57Bl6/J mice on a high-fat diet (HFD). The effects of eWAT transduction on metabolic parameters were evaluated 10 weeks later. To further define the role of LMO3 in insulin-stimulated glucose uptake, insulin signaling, adipocyte bioenergetics, as well as endocrine function, experiments were conducted in 3T3-L1 adipocytes and newly differentiated human primary mature adipocytes, engineered for transient gain or loss of LMO3 expression, respectively. AAV transduction of eWAT results in strong and stable Lmo3 expression specifically in the adipocyte fraction over a course of 10 weeks with HFD feeding. LMO3 expression in eWAT significantly improved insulin sensitivity and healthy visceral adipose tissue expansion in diet-induced obesity, paralleled by increased serum adiponectin. In vitro, LMO3 expression in 3T3-L1 adipocytes increased PPARγ transcriptional activity, insulin-stimulated GLUT4 translocation and glucose uptake, as well as mitochondrial oxidative capacity in addition to fatty acid oxidation. Mechanistically, LMO3 induced the PPARγ coregulator Ncoa1, which was required for LMO3 to enhance glucose uptake and mitochondrial oxidative gene expression. In human mature adipocytes, LMO3 overexpression promoted, while silencing of LMO3 suppressed mitochondrial oxidative capacity. LMO3 expression in visceral adipose tissue regulates multiple genes that preserve adipose tissue functionality during obesity, such as glucose metabolism, insulin sensitivity, mitochondrial function, and adiponectin secretion. Together with increased PPARγ activity and Ncoa1 expression, these gene expression changes promote insulin-induced GLUT4 translocation, glucose uptake in addition to increased mitochondrial oxidative capacity, limiting HFD-induced adipose dysfunction. These data add LMO3 as a novel regulator improving visceral adipose tissue function during obesity. KEY MESSAGES: LMO3 increases beneficial visceral adipose tissue expansion and insulin sensitivity in vivo. LMO3 increases glucose uptake and oxidative mitochondrial activity in adipocytes. LMO3 increases nuclear coactivator 1 (Ncoa1). LMO3-enhanced glucose uptake and mitochondrial gene expression requires Ncoa1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Adipocitos/metabolismo , Metabolismo Energético , Grasa Intraabdominal/metabolismo , Proteínas con Dominio LIM/genética , Obesidad/metabolismo , Células 3T3-L1 , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Biomarcadores , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Expresión Génica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Insulina/metabolismo , Grasa Intraabdominal/citología , Proteínas con Dominio LIM/metabolismo , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Modelos Biológicos , Obesidad/etiología , Oxidación-Reducción , Fosforilación Oxidativa , PPAR gamma/metabolismo , Unión ProteicaRESUMEN
In the adult mammalian skin, cells are constantly renewing, differentiating and moving upward, to finally die in a yet not fully understood manner. Here, we provide evidence that macroautophagy/autophagy has a dual role in the skin. In addition to its known catabolic protective role as an evolutionary conserved upstream regulator of lysosomal degradation, we show that autophagy induced cell death (CDA) occurs in epithelial lineage-derived organs, such as the inter-follicular epidermis, the sebaceous- and the Harderian gland. By utilizing GFP-LC3 transgenic and ATG7-deficient mice, we show that CDA is initiated during terminal differentiation at a stage when the cells have become highly resistant to apoptosis. In these transitional cells, the Golgi compartment expands, which accounts for the formation of primary lysosomes, and the nucleus starts to condense. During CDA a burst of autophagosome formation is observed, first the endoplasmic reticulum (ER) is phagocytosed followed by autophagy of the nucleus. By this selective form of cell death, most of the cytoplasmic organelles are degraded, but structural proteins remain intact. In the absence of autophagy, consequently, parts of the ER, ribosomes, and chromatin remain. A burst of autophagy was stochastically observed in single cells of the epidermis and collectively in larger areas of ductal cells, arguing for a coordinated induction. We conclude that autophagy is an integral part of cell death in keratinocyte lineage cells and participates in their terminal cell fate.Abbreviations: Atg7: autophagy related 7; BECN1: beclin 1; CDA: cell death-induced autophagy; Cre: Cre-recombinase; DAPI: 4',6-diamidino-2-phenylindole; ER: endoplasmatic reticulum; GFP: green fluorescent protein; HaGl: haderian gland; IVL: involucrin; KRT14: keratin 14; LD: lipid droplet; LSM: laser scanning microscope; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PN: perinuclear space; RB: residual body; rER: rough endoplasmatic reticulum; SB: sebum; SG-SC: stratum granulosum - stratum corneum; SGl: sebaceous gland; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labelling.
Asunto(s)
Autofagosomas/metabolismo , Autofagia/fisiología , Diferenciación Celular/fisiología , Lisosomas/metabolismo , Piel/citología , Animales , Apoptosis/fisiología , Células Epiteliales/fisiología , Ratones TransgénicosRESUMEN
No tyrosine kinase inhibitors are approved for malignant pleural mesothelioma (MPM). Preclinical studies identified focal adhesion kinase (FAK) as a target in MPM. Accordingly, we assessed the novel, highly selective FAK inhibitor (BI 853520) in 2D and 3D cultures and in vivo. IC50 values were measured by adherent cell viability assay. Cell migration and 3D growth were quantified by video microscopy and spheroid formation, respectively. Phosphorylation of FAK, Akt, S6, and Erk was measured by immunoblot. The mRNA expression of the putative tumor stem cell markers SOX2, Nanog, CD44, ALDH1, c-myc, and Oct4 was analyzed by qPCR. Cell proliferation, apoptosis, and tumor tissue microvessel density (MVD) were investigated in orthotopic MPM xenografts. In all 12 MPM cell lines, IC50 exceeded 5 µM and loss of NF2 did not correlate with sensitivity. No synergism was found with cisplatin in adherent cells. BI 853520 decreased migration in 3 out of 4 cell lines. FAK phosphorylation was reduced upon treatment but activation of Erk, Akt, or S6 remained unaffected. Nevertheless, BI 853520 inhibited spheroid growth and significantly reduced tumor weight, cell proliferation, and MVD in vivo. BI 853520 has limited effect in adherent cultures but demonstrates potent activity in spheroids and in orthotopic tumors in vivo. Based on our findings, further studies are warranted to explore the clinical utility of BI 853520 in human MPM. KEY MESSAGES: Response to FAK inhibition in MPM is independent of NF2 expression or histotype. FAK inhibition strongly interfered with MPM spheroid formation. BI 853520 has been shown to exert anti-tumor effect in MPM.
Asunto(s)
Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Mesotelioma/tratamiento farmacológico , Neoplasias Pleurales/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Neoplasias Pulmonares/patología , Mesotelioma/patología , Mesotelioma Maligno , Ratones SCID , Neoplasias Pleurales/patología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología , Células Tumorales CultivadasRESUMEN
Secretion and exchange of biomolecules via extracellular vesicles (EVs) are crucial mechanisms in intercellular communication, and the roles of EVs in infection, inflammation, or thrombosis have been increasingly recognized. EVs have emerged as central players in immune regulation and can enhance or suppress the immune response, depending on the state of donor and recipient cells. We investigated the interaction of blood cell-derived EVs with leukocyte subpopulations (monocytes and their subsets, granulocytes, B cells, T cells, and NK cells) directly in whole blood using a combination of flow cytometry, imaging flow cytometry, cell sorting, and high resolution confocal microscopy. Platelet-derived EVs constituted the majority of circulating EVs and were preferentially associated with granulocytes and monocytes, while they scarcely interacted with lymphocytes. Further flow cytometric differentiation of monocyte subsets provided clear indications for a preferential association of platelet-derived EVs with intermediate (CD14++CD16+) monocytes in whole blood.
Asunto(s)
Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Vesículas Extracelulares/metabolismo , Leucocitos/metabolismo , Biomarcadores , Plaquetas/inmunología , Separación Celular/métodos , Granulocitos/inmunología , Granulocitos/metabolismo , Humanos , Inmunofenotipificación , Leucocitos/inmunología , Monocitos/inmunología , Monocitos/metabolismoRESUMEN
Chemokines influence tumor metastasis by targeting tumor, stromal, and hematopoietic cells. Characterizing the chemokine mRNA expression profile of human primary melanoma samples, we found CXCL5 significantly up-regulated in stage T4 primary melanomas when compared to thin melanomas (T1 stage). To characterize the role of CXCL5 in melanoma progression, we established a metastasizing murine xenograft model using CXCL5-overexpressing human melanoma cells. CXCL5 had no effect on melanoma proliferation in vitro and on primary tumor growth in vivo, but CXCL5-overexpressing tumors recruited high amounts of neutrophils and exhibited significantly increased lymphangiogenesis in our severe combined immune-deficient mouse model. Recruited neutrophils were found in close proximity to or within lymphatic vessels, often in direct contact with melanoma cells. Clinically, CXCL5-overexpressing melanomas had significantly increased lymph node metastases. We were able to translate these findings to human patient samples and found a positive correlation between CXCL5 expression, numbers of neutrophils in stage T4 primary melanoma, and the occurrence of subsequent locoregional metastasis.
Asunto(s)
Quimiocina CXCL5/metabolismo , Metástasis Linfática/inmunología , Melanoma/patología , Neutrófilos/inmunología , Neoplasias Cutáneas/patología , Animales , Biomarcadores de Tumor , Comunicación Celular/inmunología , Línea Celular Tumoral , Quimiocina CXCL5/inmunología , Femenino , Estudios de Seguimiento , Humanos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Linfangiogénesis/inmunología , Metástasis Linfática/patología , Melanoma/inmunología , Ratones , Ratones Pelados , Ratones SCID , Estadificación de Neoplasias , Neutrófilos/metabolismo , ARN Mensajero/metabolismo , Neoplasias Cutáneas/inmunología , Organismos Libres de Patógenos Específicos , Esferoides Celulares , Regulación hacia ArribaRESUMEN
Purpose: Malignant pleural mesothelioma (MPM) is an aggressive thoracic tumor type with limited treatment options and poor prognosis. The angiokinase inhibitor nintedanib has shown promising activity in the LUME-Meso phase II MPM trial and thus is currently being evaluated in the confirmatory LUME-Meso phase III trial. However, the anti-MPM potential of nintedanib has not been studied in the preclinical setting.Experimental Design: We have examined the antineoplastic activity of nintedanib in various in vitro and in vivo models of human MPM.Results: Nintedanib's target receptors were (co)expressed in all the 20 investigated human MPM cell lines. Nintedanib inhibited MPM cell growth in both short- and long-term viability assays. Reduced MPM cell proliferation and migration and the inhibition of Erk1/2 phosphorylation were also observed upon nintedanib treatment in vitro Additive effects on cell viability were detected when nintedanib was combined with cisplatin, a drug routinely used for systemic MPM therapy. In an orthotopic mouse model of human MPM, survival of animals receiving nintedanib per os showed a favorable trend, but no significant benefit. Nintedanib significantly reduced tumor burden and vascularization and prolonged the survival of mice when it was administered intraperitoneally. Importantly, unlike bevacizumab, nintedanib demonstrated significant in vivo antivascular and antitumor potential independently of baseline VEGF-A levels.Conclusions: Nintedanib exerts significant antitumor activity in MPM both in vitro and in vivo These data provide preclinical support for the concept of LUME-Meso trials evaluating nintedanib in patients with unresectable MPM. Clin Cancer Res; 24(15); 3729-40. ©2018 AACR.
Asunto(s)
Indoles/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Mesotelioma/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Neoplasias Pleurales/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mesotelioma/genética , Mesotelioma/patología , Mesotelioma Maligno , Ratones , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Fosforilación/efectos de los fármacos , Neoplasias Pleurales/genética , Neoplasias Pleurales/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of ±4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.
Asunto(s)
Inmunohistoquímica/métodos , Fibras Musculares Esqueléticas/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Animales , Automatización , Núcleo Celular/metabolismo , Fibras Musculares Esqueléticas/citología , Ratas , Coloración y Etiquetado , Factores de TiempoRESUMEN
The oncogenic potential of the transcriptional repressor Bcl-6 (B-cell lymphoma 6) was originally discovered in non-Hodgkin patients and the soluble Bcl-6 inhibitor 79-6 was developed to treat diffuse large B-cell lymphomas with aberrant Bcl-6 expression. Since we found Bcl-6 and its co-repressor BCoR (Bcl-6 interacting co-repressor) to be regulated in human microvascular endothelium by colorectal cancer cells, we investigated their function in sprouting angiogenesis which is central to tumor growth. Based on Bcl-6/BCoR gene silencing we found that the transcriptional repressor complex in fact constitutes an endogenous inhibitor of vascular sprouting by supporting the stalk cell phenotype: control of Notch target genes (HES1, HEY1, DLL4) and cell cycle regulators (cyclin A and B1). Thus, when endothelial cells were transiently transfected with Bcl-6 and/or BCoR siRNA, vascular sprouting was prominently induced. Comparably, when the soluble Bcl-6 inhibitor 79-6 was applied in the mouse retina model of physiological angiogenesis, endothelial sprouting and branching were significantly enhanced. To address the question whether clinical treatment with 79-6 might therefore have detrimental therapeutic effects by promoting tumor angiogenesis, mouse xenograft models of colorectal cancer and diffuse large B-cell lymphoma were tested. Despite a tendency to increased tumor vessel density, 79-6 therapy did not enhance tumor expansion. In contrast, growth of colorectal carcinomas was significantly reduced which is likely due to a combined 79-6 effect on cancer cells and tumor stroma. These findings may provide valuable information regarding the future clinical development of Bcl-6 inhibitors.